A lipid bilayer membrane is a basic structure of a biological membrane, and is found in a biological membrane of every living organism. In most of biological membranes, various membrane proteins are provided in the lipid bilayer membrane, and the membrane protein is involved in (i) transportation of substances such as various types of ion, sugar, amino acid, nucleotide, and the like, (ii) transmission of signals, or (iii) synthesis of lipid.
In this way, the biological membrane is a place for exhibiting vital functions and plays various important roles such as recognition of external information, transmission of the information into the membrane, conversion of the substance, transportation of the substance, and the like. Further, in case of applying functions of the biological membrane, various industrial applicability can be found. Thus, it is extremely important to produce an artificial membrane as a biological membrane model.
As the artificial membrane serving as the biological membrane model, various membranes such as a polymer cast membrane, a Langmuir-Blodigett (LB) membrane, and the like are used, but an artificial lipid bilayer membrane is most similar to the biological membrane. The artificial lipid bilayer membrane is a thin membrane in which amphipathic molecules are aligned so that alkyl chains of hydrophobic portions of the amphipathic molecules internally face each other. For example, the artificial lipid bilayer membrane is applied to measurement of an ion current of an ion channel and a similar use.
Incidentally, as a method for forming the artificial lipid bilayer membrane, the planar lipid bilayer method is known. As a method for measuring an ion current of a single ion channel, a patch-clamp method is adopted. However, in order to more deeply study correlation of structural functions of the channel, it is necessary to use a simple rearrangement system in carrying out an experiment. An artificial lipid bilayer membrane formation method adopted in this case is the planar lipid bilayer method.
In the planar lipid bilayer method, a minimum simple system including ion, water, an artificial lipid bilayer membrane, and an ion channel is used so as to study a basic structure of the ion channel and detail correlation of structural functions thereof (Non-Patent Document 1).
The following specifically explains a system of the planar lipid bilayer method. As illustrated in FIG. 13, an ion channel 112 is provided in an artificial lipid bilayer membrane 111, and a current flowing via the ion channel 112 is measured. The artificial lipid bilayer membrane 111 is formed on a small hole 115 provided in a partition plate 114 such as a plastic plate for parting an aqueous solution chamber 113. In one of two chambers obtained by parting the aqueous solution chamber 113, an electrode 116 is provided. Via the electrode 116, a current measuring device 117 is provided. In the other chamber, an electrode 118 is provided. Via the electrode 118, an earth 119 causes the aqueous solution chamber 113 to be earthed.
Here, examples of how to form the artificial lipid bilayer membrane 111 on the small hole 115 include (A) vertical painting method, (B) vertical applying method, (C) horizontal formation method, and the like.
In the (A) vertical painting method, first, as illustrated in the left illustration of FIG. 14, with a thin glass tube or the like, lipid solution 110 is applied to the small hole 115 provided in a support such as the plate 114 for parting the aqueous solution chamber 113 (not shown in FIG. 14). Under this condition, the lipid solution 110 swells in directions of both surfaces of the partition plate 114 so as to cover the small hole 115. The lipid solution 110 is obtained by dissolving lipid in organic solvent such as decane. After applying the lipid solution 110, the lipid solution 110 moves on the surface of the plate 114 as illustrated in the right illustration of FIG. 14, thereby obtaining an artificial lipid bilayer membrane which has become thinner in a natural manner. Note that, the wording “become thinner” means a process in which the organic solvent or the like moves from a central portion of the applied lipid solution 110 so that a lipid bilayer membrane is formed in the central portion.
Next, in the (B) vertical applying method, as illustrated in the upper illustration of FIG. 15, a lipid monomolecular membrane 121 are developed on a gas-liquid interface of the aqueous solution chamber 113 (not shown in FIG. 15). At this time, a position of the gas-liquid interface is the same as a position of a lower side end of the small hole 115 provided in the partition plate 114. Thereafter, as illustrated in the middle illustration of FIG. 15, a liquid surface (gas-liquid interface) of one chamber (right side of the middle illustration) of two chambers obtained by parting the aqueous solution chamber 113 is raised, thereby developing the monomolecular membrane 121 on the surface of the partition plate 114. On this account, one opening side of the small hole 115 is covered by the monomolecular membrane 121. Thereafter, as illustrated in the lower illustration of FIG. 15, a liquid surface (gas-liquid interface) of the other chamber (left side of the lower illustration) of two chambers obtained by parting the aqueous solution chamber 113 is raised, thereby developing the monomolecular membranes 121 on the surface of the partition plate 114. On this account, also the other opening side of the small hole 115 is covered by the monomolecular membrane 121. As a result, on each opening side of the small hole 115, the monomolecular membrane 121 is applied, so that the artificial lipid bilayer membrane 111 is finally formed.
Next, in the (C) horizontal formation method, the aqueous solution chamber 113 illustrated in FIG. 13 is vertically parted with the partition plate 114. At this time, as illustrated in FIG. 16(a), the small hole 115 provided in the partition plate 114 is covered by the lipid solution 110, and the lipid solution 110 is left until the lipid solution 110 becomes thinner in a natural manner as the artificial lipid bilayer membrane 111. Alternatively, as illustrated in FIG. 16(b), a hydraulic pressure above the small hole 115 is raised in the chamber so that the lipid solution 110 extends downward so as to be thinner, thereby forming the artificial lipid bilayer membrane 111.
However, in any one of the artificial lipid bilayer membrane formation methods, it is difficult to quickly form a stable artificial lipid bilayer membrane 111. That is, in the (A) vertical painting method, it takes several minutes to dozens minutes for the lipid solution 110 to move on the surface of the partition plate 114 and become sufficiently thinner as the artificial lipid bilayer membrane 111. Further, in the (B) vertical applying method, it is essential to carry out a pre-treatment with respect to the small hole 115 with organic solvent such as squalene before forming the artificial lipid bilayer membrane 111, so that such a larger number of steps results in a more complicate formation method. Further, it is general that the artificial lipid bilayer membrane 111 is not formed unless the liquid surface is raised and lowered several times.
Further, in the (C) horizontal formation method, in case of leaving the lipid solution 110 covering the small hole 115 until the lipid solution 110 becomes thinner in a natural manner (in case of FIG. 16(a)), it is impossible to intentionally control the formation of the artificial lipid bilayer membrane. Therefore, it sometimes takes several hours for the lipid solution 110 to become thinner. Further, in case of raising the hydraulic pressure above the small hole 115 in the chamber so that the lipid solution 110 becomes thinner (in case of FIG. 16(b)), the obtained artificial lipid bilayer membrane 111 has a thin portion serving as the “lipid bilayer membrane” and a thick portion referred to as a bulk layer surrounding the thin portion. In this manner, the artificial lipid bilayer membrane 111 obtained in this method is based on physicochemical balance of the foregoing portions. Thus, if these portions are physicochemically unbalanced by vibration caused by aqueous solution flow or the like, the artificial lipid bilayer membrane 111 is easily broken. Moreover, it is difficult to exactly control a pressure difference between the upper and lower chambers of the aqueous solution chamber 113, so that the obtained artificial lipid bilayer membrane 111 is likely to be unstable.
In case of adopting the planar lipid bilayer method, it is necessary to realize a great object: to form a stable and durable artificial lipid bilayer membrane.
The inventors of the present invention proposed a technique for solving the conventional problems in the artificial lipid bilayer membrane used in a current measuring device (for example, Non-Patent Document 2). In the current measuring device obtained by this technique, it is possible to simultaneously measure both a structure and a function of ion channel molecules by using the artificial lipid bilayer membrane.
Specifically, as illustrated in FIG. 17, the current measuring device includes two solution chambers: an upper solution chamber 101 and a lower solution chamber 102. On a central portion of a bottom of the upper solution chamber 101, a film 103 having a small hole 105 is applied. Further, the lower solution chamber 102 has an opening 104 in its bottom, and a cover glass 106 is fixed on the opening 104 with an adhesive. On the cover glass 106, an agarose gel layer (not shown) is formed.
In the current measuring device, first, a lower portion of the upper solution chamber 101 is moved in the lipid solution so as to form a thick membrane made of lipid solution in the small hole 105. Thereafter, the upper solution chamber 101 is placed in the lower solution chamber 102, and the upper solution chamber 101 is lowered so that the thick membrane formed in the small hole 105 comes into contact with the agarose gel layer formed on the cover glass 106. Here, the pressure (hydraulic pressure) in the upper solution chamber 101 is raised so that surplus lipid solution is extruded from a gap of the agarose gel layer, so that an artificial lipid bilayer membrane is formed by making the thick membrane thinner.
In the current measuring device, the pressure in the upper solution chamber 101 is raised, so that it takes less time to form an artificial lipid bilayer membrane (to make the thin membrane thinner). The thus formed artificial lipid bilayer membrane is supported by the agarose gel layer. Thus, even when a pressure is exerted by the upper solution chamber 101, the artificial lipid bilayer membrane is stabilized in upward and downward directions.
[Non-Patent Document 1]
“New Patch-Clamp Test” written by Shigetoshi Oiki, published by Yoshioka-shoten, 2001, pages 208-215, “19. planar lipid bilayer method for Studying Channel”
[Non-Patent Document 2]
Ide, T., Takeuchi, U., Yanagida, T. Development of an Experimental Apparatus for Simultaneous Observation of Optical and Electrical Signals from Single Ion Channels, Single Mol. 3(2002) 1, pages 33-42
However, in the conventional technique for forming the artificial lipid bilayer membrane, it is sometimes difficult to stably form the artificial lipid bilayer membrane, so that a more stable formation technique is required.
Specifically, in the conventional technique, the pressure (hydraulic pressure) in the upper solution chamber 101 is raised so that surplus lipid solution is extruded from a gap of the agarose gel layer, thereby making the thick membrane thinner. At this time, when the pressure of the upper solution chamber 101 is raised, as illustrated in FIG. 18(a), the surplus lipid moves away in R directions between the bottom of the upper solution chamber 101 and the agarose gel layer 108, so that it is possible to make the thick membrane thinner. However, as illustrated in FIG. 18(b), a higher pressure in the upper solution chamber 101 allows the aqueous solution to move from the upper solution chamber 101 to the lower solution chamber 102 (in M directions). Thus, as illustrated in FIG. 18(c), the artificial lipid bilayer membrane 111 having been made thinner is likely to excessively expand and is likely to be broken.
In case where the thick membrane made of lipid solution so as to cover the small hole is made thinner in this manner, when a technique of raising the pressure in the upper solution chamber is adopted, there occurs such problem that it is impossible to make the thick membrane thinner in a stable manner and the artificial lipid bilayer membrane is likely to be broken.